Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
  • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
  • C12N9/6424—Serine endopeptidases (3.4.21)
  • C12N9/6437—Coagulation factor VIIa (3.4.21.21)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/36—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood coagulation factors
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
  • C12Y304/21—Serine endopeptidases (3.4.21)
  • C12Y304/21021—Coagulation factor VIIa (3.4.21.21)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
  • Y10S530/806—Antigenic peptides or proteins
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
  • Y10S530/807—Hapten conjugated with peptide or protein
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
  • Y10S530/81—Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
  • Y10S530/81—Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier
  • Y10S530/811—Peptides or proteins is immobilized on, or in, an inorganic carrier
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
  • Y10S530/81—Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier
  • Y10S530/812—Peptides or proteins is immobilized on, or in, an organic carrier

Definitions

• the invention relates to synthetic peptides which contain certain partial sequences from factor VIIa, their synthesis and the use of these peptides for immunizing an animal and for the purification of specific antibodies against the said peptides, to antibodies against these peptides and the use of these antibodies and peptides in therapy and diagnostics.
• the determination of the functional activity of factor VII using factor VII deficient plasma is known (John T. Brandt et al., Am.J. Clin. Pathol. 85 (1986), 583-589).
• the coagulation time reducing effect of diluted plasma is determined in a system which contains all the factors necessary for the coagulation process with the exception of factor VII.
• the test completely detects the fraction of factor VII which can be activated in the plasma sample, but is unable to determine the concentration of active factor VIIa which is already present.
• a method for the quantification of factor VII using radio or enzyme immunoassays is also known (C. Bayer et al., Thrombosis and Haemostasis, 56 (3) (1986), 250-255).
• the antibodies required for this are generated by using factor VII purified from plasma to immunize animals.
• the antibodies obtained are suitable for the quantitative determination of factor VII, but do not make it possible to differentiate between inactive factor VII and active factor VIIa.
• the object of the present invention was therefore to provide antigens which lead to the formation of specific antibodies against factor VIIa. Furthermore, the task was based on the development of a test method using specific factor VIIa antibodies, which enables a sensitive and exact quantification of factor VIIa or factor VIIa / TF complex in biological liquids.
• the invention therefore relates to peptides which have amino acid sequences which partially correspond to the carboxy- or amino-terminal ends of factor VIIa formed by factor Xa cleavage of factor VII, characterized in that they have the amino acid sequence H-Ser-Asp-His- Thr-Gly-Thr-Lys-Arg-Ser-Cys-Arg-Cys-His-Glu-Gly-Tyr-Ser-Leu-Leu-Ala-Asp-Gly-Val-Ser-Cys-Thr-Pro-Thr- Val-Glu-Tyr-Pro-Cys-Gly-Lys-Ile-Pro-Ile-Leu-Glu-Lys-Arg-Asn-Ala-Ser-Lys-Pro-Gln-Gly-Arg-OH and / or the sequence H-Ile-Val-Gly-Gly-Lys-Val-Cys-Pro-Lys-Gly-Glu-Cys-Pro
• the invention further relates to the use of the peptides according to the invention for the production of antibodies, the production of antibodies by immunoadsorptive purification from polyclonal antisera being preferred.
• the invention also relates to the use of the antibodies according to the invention and / or the peptides according to the invention for determining factor VIIa or factor VIIa / TF complex.
• the Fmoc group, the permanent protective groups for the side functions based on t-butyl / Boc, for Arg the Pmc or Mtr group and for Cys the tert-butyl mercapto groups or trityl groups are preferably used as the temporary protective group.
• the C-terminal amino acid is immobilized via p-alkoxybenzyl ester groups which are bound to a polymeric carrier which is usually suitable for peptide synthesis, preferably crosslinked polystyrene.
• Any sulfhydryl groups to be exposed are "deprotected" with tri-n-butylphosphine in an alcohol, for example trifluoroethanol or with DTT in water.
• an alcohol for example trifluoroethanol or with DTT in water.
• Cys (Trt) deprotection a separate work step when using ethanedithiol as a scavenger is unnecessary.
• the purification of the peptides can e.g. B. done via ion exchange chromatography, reversed-phase chromatography and gel permeation chromatography. The correct composition of the peptides and the peptide contents are determined by amino acid analysis.
• the peptides In view of the intended use for the peptides, it makes sense to introduce amino acids with reactive side groups into the peptides in such a way that they do not influence the structure of the hapten. For this reason, cysteine is expediently added at the N or C terminal, the free SH group of which is suitable for coupling to many carriers via thioether. Is preferably z.
• the antigen represented by the above-mentioned peptide is provided in the form of the decapeptide Cys-Arg-Asn-Ala-Ser-Lys-Pro-Gln-Gly-Arg.
• the peptide used for immunization can be produced either in a manner known per se to the person skilled in the art by chemical synthesis, or by purification of a polypeptide provided by genetic engineering.
• Peptides that are to be used for immunization or those that are to be used as an immunoadsorbent are expediently coupled to a carrier molecule. Coupling methods are known per se to the person skilled in the art and are described in the literature (Nakane, PK et al., J. Histochem. Cytochem. 22 (1974), 1084-1091).
• Carrier molecules in the sense of this invention can be natural or synthetic macromolecules as they are Those skilled in the art used to generate an immunoreactive conjugate, such as. B. albumin, ovalbumin, keyhole limpet hemocyanin or polysaccharides.
• the peptide or polypeptide is bound to the hemocyanin of a marine limpet, the keyhole limpet hemocyanin.
• Carrier molecules in this sense are insoluble polymers, as used by those skilled in the art for the immobilization of proteins and peptides, such as. B. polystyrene, nylon, agarose or magnetizable particles.
• the solid phase can be in any form, for. B. present as a tube, fleece, ball or microparticles.
• a preferred embodiment provides the coupling of peptides, e.g. B. the above-mentioned decapeptides, on bromocyan-activated Sepharose.
• the immunoglobulin fraction relevant for specific tests can be enriched by conventional immunoadsorptive methods.
• That to the immunadorptive Peptide used for purification can also have a truncated amino acid sequence; The only requirement for use in immunoadsorptive purification of the desired antibody is that the antigenic determinant formed by this shorter polypeptide is recognized by the desired antibody and effectively bound.
• the peptide used for the immunoadsorptive production of the antibodies can e.g. B. be a decapeptide, preferably the peptide Cys-Arg-Asn-Ala-Ser-Lys-Pro-Gln-Gly-Arg.
• antibodies are induced in the animal system by immunization with synthetic peptides and purified by immunoadsorption. These antibodies react specifically with the peptides used for immunization and purification. Depending on the sequence of the peptide used, these antibodies either bind only to factor VIIa or, if a peptide sequence exposed in the native factor VII molecule is selected, to the intact factor VII molecule.
• antibodies can be selected which react specifically with the antigenic determinants of factor VIIa, which correspond to the recognition sequence of the factor Xa cleavage site of this molecule.
• peptides that have the C- or N-terminal factor Xa recognition sequence are used both for immunization and for immunoadsorptive purification, antibodies against these sequences are enriched.
• these do not react with intact, native factor VII, since the factor Xa cleavage site in the intact factor VII molecule is either not exposed sufficiently or does not have the higher structure required for antigenic recognition.
• the antibodies obtained according to the invention can be used for homogeneous and heterogeneous immunoassays known to those skilled in the art, such as, for. B. enzyme immunoassays or free or latex-enhanced agglutination reactions.
• they are preferably coupled to a solid support.
• solid supports are known per se to the person skilled in the art, such as, for. B. microtitration plates, tubes, balls, microbeads, magnetizable particles and.
• the immobilization on polystyrene tubes or microtitration plates is preferred.
• the tubes prepared for the following immunoassays can then be sealed airtight, e.g. B. stored at 4 ° C.
• the determination of the content of factor VIIa according to the invention is carried out by pre-incubating the sample with such immobilized antibodies, the concentration of factor VIIa bound by the immobilized antibodies being detected by a subsequent incubation with a second antibody.
• This second antibody must have a property that is measurable, e.g. B. the ability to convert or bind a chromogenic substrate.
• the second antibody can e.g. B. with an enzyme, a fluorescent molecule, such as. B. fluorescein isothiocyanate, a radioactive label or a molecule capable of chemiluminescence.
• This second antibody is preferably coupled to a marker enzyme; peroxidase is particularly preferred.
• the concentration of factor VIIa / TF complex can also be determined with an antibody immobilized in this way. Use is a prerequisite of a specific antibody against tissue factor as a second antibody, which is labeled in the manner described.
• TF antibodies can be obtained as polyclonal or monoclonal antibodies by methods known to those skilled in the art. The TF antibody can also be bound and the factor VIIa antibody labeled.
• Factor VIIa or Factor VIIa / TF complex can also be determined by simultaneous incubation of the sample, preferably plasma and labeled antibody, with the immobilized antibodies.
• a competitive determination method is possible, where labeled and unlabelled factor VIIa or factor VIIa / TF complex compete for the binding site of the immobilized antibodies.
• the factor VIIa content determined in this way allows a statement about the degree of activation of factor VII.
• the embodiments given in the examples are particularly preferred.
• Plasma anticoagulated with citrate solution was mixed with thromboplastin solution and incubated at 37 ° C. Aliquots were removed at various times and the reaction was stopped by adding citrate (final concentration: 0.15 mol / L). The samples were diluted with incubation buffer 1 + 1 and tested with the ELISA.
• the peptides according to the invention which have an amino acid sequence which corresponds wholly or partly to the amino acid sequence of factor VII and are antigen thus induce the binding-specific antibodies against the respective antigenic determinants present in the peptide. These specific antibodies can then be purified by immunoadsorption on peptides with the same antigenic determinant.
• the use of synthetic peptides has the essential advantage that absolute pure antigens are used for immunization, so that no cross-reaction with other proteins or other parts of the factor VII molecule can occur in the resulting antiserum.
• a peptide which corresponds to the C- or N-terminal amino acid sequence of the factor Xa cleavage site in the factor VII molecule is preferably used.
• Factor VIIa With an antibody against this peptide, only cleaved factor VII molecules can be detected, i.e. Factor VIIa, since in the intact native factor VII this sequence of antibody detection is not accessible.
• the measurement of the amount of bound antibody by means of ELISA allows a direct conclusion to be drawn about the concentration of factor VIIa or factor VIIa-TF complex formed and thus a statement about the degree of activation of factor VII.

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• 1990
  • 1990-03-13 DE DE4007902A patent/DE4007902A1/de not_active Withdrawn
• 1991
  • 1991-03-08 AT AT91103555T patent/ATE193707T1/de not_active IP Right Cessation
  • 1991-03-08 EP EP91103555A patent/EP0446797B1/fr not_active Expired - Lifetime
  • 1991-03-08 DE DE59109189T patent/DE59109189D1/de not_active Expired - Lifetime
  • 1991-03-08 ES ES91103555T patent/ES2149155T3/es not_active Expired - Lifetime
  • 1991-03-11 US US07/666,913 patent/US5254672A/en not_active Expired - Lifetime
  • 1991-03-11 AU AU72758/91A patent/AU653332B2/en not_active Ceased
  • 1991-03-12 CA CA002038030A patent/CA2038030C/fr not_active Expired - Fee Related
  • 1991-03-12 JP JP3070418A patent/JPH04217996A/ja active Pending

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